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Liquid phase separation of proteins based on electrophoretic effects in an electrospray setup during sample introduction into a gas-phase electrophoretic mobility molecular analyzer (CE-GEMMA/CE-ES-DMA).

Weiss VU, Kerul L, Kallinger P, Szymanski WW, Marchetti-Deschmann M, Allmaier G - Anal. Chim. Acta (2014)

Bottom Line: This makes the EM determination of individual species sometimes difficult, if not impossible.This finding was consecutively applied for on-line desalting allowing EM diameter determination of analytes despite a high salt concentration within samples.Results demonstrate the proof of concept of such an approach and additionally illustrate the high potential of a future on-line coupling of a capillary electrophoresis to a GEMMA instrument.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria.

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Comparison of the GEMMA spectra obtained for a BSA and IgG containing sample with and without sodium chloride addition in NM and CM: samples containing both analytes, BSA and IgG at c = 1 μmol L−1 (pH 9.4 ammonium acetate) were measured in NM (A); solid lines represent samples without salt addition, dashed lines depict the samples with sodium chloride addition (c = 5 mmol L−1). The salt addition also increases the background signal for a sample containing no protein, i.e., plain ammonium acetate (B). Additionally, BSA can no longer be detected in the presence of sodium chloride, the EM diameter of IgG is shifted to higher values and the width of the IgG peak increases significantly. Samples containing both analytes at c = 1 μmol L−1 and sodium chloride at c = 5 mmol L−1 were measured in CM as well (C) as were buffer blanks (D). The EM diameter of BSA and IgG correlates with the values determined from samples without sodium chloride addition measured in NM (see Fig. 5). Analytes were separated from the contaminating salt. Measurement conditions as in Fig. 3.
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fig0030: Comparison of the GEMMA spectra obtained for a BSA and IgG containing sample with and without sodium chloride addition in NM and CM: samples containing both analytes, BSA and IgG at c = 1 μmol L−1 (pH 9.4 ammonium acetate) were measured in NM (A); solid lines represent samples without salt addition, dashed lines depict the samples with sodium chloride addition (c = 5 mmol L−1). The salt addition also increases the background signal for a sample containing no protein, i.e., plain ammonium acetate (B). Additionally, BSA can no longer be detected in the presence of sodium chloride, the EM diameter of IgG is shifted to higher values and the width of the IgG peak increases significantly. Samples containing both analytes at c = 1 μmol L−1 and sodium chloride at c = 5 mmol L−1 were measured in CM as well (C) as were buffer blanks (D). The EM diameter of BSA and IgG correlates with the values determined from samples without sodium chloride addition measured in NM (see Fig. 5). Analytes were separated from the contaminating salt. Measurement conditions as in Fig. 3.

Mentions: Results from on-line desalting in the nano ES capillary of the GEMMA instrument (addition of 5 mmol L−1 sodium chloride to a mixed BSA/IgG sample in ammonium acetate) are depicted in Fig. 6, together with results for the same sample obtained in NM. GEMMA experiments in NM exhibit results shown in Fig. 6(A) (for samples) and Fig. 6(B) (for blanks), dashed lines, respectively. For comparison reasons also spectra recorded in the absence of sodium chloride (solid lines) are shown. Addition of sodium chloride leads to a massive increase in the background signal (Fig. 6(B)) due to salt aggregation in droplets generated by the nano ES process and their subsequent detection as residue particles. Concurrently, also aggregates between proteins and salt molecules are recorded. Whereas the IgG peak maximum shifts to higher EM diameter values by about 6% due to an unwanted salt crust formation around the protein, BSA can be no longer detected due to the increased background signal. Additionally, the IgG peak also increases in peak width as a result from heterogeneous protein/salt aggregates.


Liquid phase separation of proteins based on electrophoretic effects in an electrospray setup during sample introduction into a gas-phase electrophoretic mobility molecular analyzer (CE-GEMMA/CE-ES-DMA).

Weiss VU, Kerul L, Kallinger P, Szymanski WW, Marchetti-Deschmann M, Allmaier G - Anal. Chim. Acta (2014)

Comparison of the GEMMA spectra obtained for a BSA and IgG containing sample with and without sodium chloride addition in NM and CM: samples containing both analytes, BSA and IgG at c = 1 μmol L−1 (pH 9.4 ammonium acetate) were measured in NM (A); solid lines represent samples without salt addition, dashed lines depict the samples with sodium chloride addition (c = 5 mmol L−1). The salt addition also increases the background signal for a sample containing no protein, i.e., plain ammonium acetate (B). Additionally, BSA can no longer be detected in the presence of sodium chloride, the EM diameter of IgG is shifted to higher values and the width of the IgG peak increases significantly. Samples containing both analytes at c = 1 μmol L−1 and sodium chloride at c = 5 mmol L−1 were measured in CM as well (C) as were buffer blanks (D). The EM diameter of BSA and IgG correlates with the values determined from samples without sodium chloride addition measured in NM (see Fig. 5). Analytes were separated from the contaminating salt. Measurement conditions as in Fig. 3.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4150488&req=5

fig0030: Comparison of the GEMMA spectra obtained for a BSA and IgG containing sample with and without sodium chloride addition in NM and CM: samples containing both analytes, BSA and IgG at c = 1 μmol L−1 (pH 9.4 ammonium acetate) were measured in NM (A); solid lines represent samples without salt addition, dashed lines depict the samples with sodium chloride addition (c = 5 mmol L−1). The salt addition also increases the background signal for a sample containing no protein, i.e., plain ammonium acetate (B). Additionally, BSA can no longer be detected in the presence of sodium chloride, the EM diameter of IgG is shifted to higher values and the width of the IgG peak increases significantly. Samples containing both analytes at c = 1 μmol L−1 and sodium chloride at c = 5 mmol L−1 were measured in CM as well (C) as were buffer blanks (D). The EM diameter of BSA and IgG correlates with the values determined from samples without sodium chloride addition measured in NM (see Fig. 5). Analytes were separated from the contaminating salt. Measurement conditions as in Fig. 3.
Mentions: Results from on-line desalting in the nano ES capillary of the GEMMA instrument (addition of 5 mmol L−1 sodium chloride to a mixed BSA/IgG sample in ammonium acetate) are depicted in Fig. 6, together with results for the same sample obtained in NM. GEMMA experiments in NM exhibit results shown in Fig. 6(A) (for samples) and Fig. 6(B) (for blanks), dashed lines, respectively. For comparison reasons also spectra recorded in the absence of sodium chloride (solid lines) are shown. Addition of sodium chloride leads to a massive increase in the background signal (Fig. 6(B)) due to salt aggregation in droplets generated by the nano ES process and their subsequent detection as residue particles. Concurrently, also aggregates between proteins and salt molecules are recorded. Whereas the IgG peak maximum shifts to higher EM diameter values by about 6% due to an unwanted salt crust formation around the protein, BSA can be no longer detected due to the increased background signal. Additionally, the IgG peak also increases in peak width as a result from heterogeneous protein/salt aggregates.

Bottom Line: This makes the EM determination of individual species sometimes difficult, if not impossible.This finding was consecutively applied for on-line desalting allowing EM diameter determination of analytes despite a high salt concentration within samples.Results demonstrate the proof of concept of such an approach and additionally illustrate the high potential of a future on-line coupling of a capillary electrophoresis to a GEMMA instrument.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria.

Show MeSH
Related in: MedlinePlus